A Rapid Molecular Method for Detection of Spoilage Yeasts in Orange Juice

Yeasts can survive in high acid and low pH orange juice and result in spoilage, but the routine detection method for yeasts, plate counting technique, is labor intensive and time-consuming. The detection is hysteretic and can't satisfy the need of yeast control for the orange juice industry. The detection limit of regular PCR could not satisfy the need of detecting yeast at low levels as 10(1)-10(0) cfu/ml, and qPCR has higher instrument and technology requirements. To meet the needs of custom quarantine and market detection, a rapid molecular method for detection of spoilage yeasts in orange juice was developed in the present study. To extract yeast DNA from orange juice, a modified rapid DNA extraction method was used, by which more than 90 mu g DNA was extracted from 1.5 ml 10(7) yeast culture. The main points of the method were using plastic pestle to grind the cell pellet by hand for 3min and diluting the DNA before use. The DNA templates were tested by PCR using yeast universal primer NL1 and NL4. With this method, yeast at a low level of 10(1) cfu/ml in orange juice and 10(0) cfu/ml in water could be detected and the whole detection procedure can be finished within 5 h.